1. Field of the Invention
The present invention relates to an anti-cancer bispecific antibody constructed by gene engineering, nucleotide sequences encoding the said bispecific antibody, the expression vectors containing the said nucleotide sequences and host cells containing the vectors.
2. Description of the Related Art
Different from natural antibodies, two antigen-binding sites of bispecific antibody (BsAb) bear different specificities, therefore, it is bivalent in chemical structure but monovalent in binding function. BsAb directed to both tumor-associated antigens and trigger molecules on effector cells can recruit the immunological effector cells to tumor sites efficiently and activate them to kill tumor cells specifically. BsAbs are hybrid proteins that can be generated by chemical cross-link, hybridoma technology or genetic methods. In the chemical cross-link method, two kinds of monoclonal antibodies and fragments thereof were dissociated by reductants to generate monovalent antibodies and fragments thereof. The resulting BsAb is constructed via chemical cross-linking of two monovalent antibodies and fragments thereof from different parental antibodies. This strategy can be used for rapid production of BsAb in large scale but BsAb can be inactivated sometimes during cross-link and it is difficult to guarantee the homogeneity of products. Another strategy for production of BsAb is hybridoma technology by which an established hybridoma cell line secreting one monoclonal antibody was fused to spleen cells immunized with the other antigen or two established hybridoma cell lines secreting two different monoclonal antibodies were fused each other to create hybrid hybridomas. The former resulting hybridoma is called dimeric hybridoma and tetrameric hybridoma. Generally, BsAb produced by hybridoma technology keeps high bioactivities. However, the procedures are tedious and time-consuming and it is not easy to isolate BsAb from other non-active and unwanted antibodies generated simultaneously. These BsAb formats encountered another predictable problems: too large size and murine components contained in BsAb are immunogenic in patients and will induce the production of human anti-mouse antibodies (HAMA), which may prevent reuse of these BsAbs in clinic. Furthermore, production and purification of these formats of BsAb are expensive, which limits the application of BsAbs in clinic. Replacement of these traditional methods with gene recombination approaches has accelerated progress in this area. Based on the technology of small molecular antibodies, production of BsAb by gene engineering has advantages over those described above, such as the stability of process, large scale production, low cost and easy-to-use. Gene engineering has led to the development of various small molecular BsAb formats by connecting two different kinds of scFvs. There are three kinds of BsAb formats classified by different links. (1) mini-antibodies are heterodimers assembled by connecting two scFv fragments together with an oligomerized domain (e.g. leucine zipper motifs derived from Fos or Jun transcription factors). (2) Diabodies are non-covalently associated dimmers which are assembled by two single chains VH1-VL2 and VH2-VL1, both connected by a short linker that is too short to allow pairing between V-domains from the same chain. Thus, each chain alone is not capable of binding antigen, but co-expression of two chains (VH1-VL2 and VH2-VL1) leads to assembly of heterodimeric diabodies which can bind to two kinds of antigens. (3) ScBsAb: a interlinker was used for connecting two different scFvs with different specificities and ScBsAb was expressed in the host cells as a single polypeptide. The intralinker between two domains within scFv is often (Gly4Ser)3. As for the interlinker between two scFvs, there are two strategies for designing it. For the purpose of avoiding false paring between heterogenous variable regions, the interlinker is often a short peptide linker less than ten amino acid residues such as Gly4Ser. Another strategy is to select a longer linker for the interlinker. In our lab, an interlinker with 25 amino acids named 205c′, devised by Gruber in construction anti-TCR×anti-fluorescent scBsAb, was cited for one of three interlinkers. Another two interlinkers named Fc (26 residues) and HSA were devised, which both result in the proper folding of two scFvs and the formation of BsAb with two antigen-binding sites with high activities. In a word, the most important for designing interlinkers is to ensure the proper pairing between variable domains and folding of proteins, resulting in the formation of BsAb which maintains biological activities and stability. Some novel properties for facilitating purification and extending the plasma half-life time should be introduced.
BsAb-mediated immunotherapy plays a promising role in the clinical biotherapy for tumors. The following is two characteristics of BsAb. First, tumor-killing effects mediated by BsAb are based on stimulating the immune system, highly specific with tumors and free of MHC restriction. Second, due to lacking Fc domain, BsAb is harmless to normal tissues. Therefore, BsAb-mediated therapy is the complementarity of traditional methods such as surgery, radiotherapy and chemotherapy. The major effect of this approach is based on clearing up sub-clinical residuals and preventing or eliminating the tumor from recurrence and metastasis. BsAb can not only cure tumors but also stimulate the immune system to provide and keep the immune protection for a long time. Based on results of experiments in mouse and clinic, BsAb prepared for trial use should have at least five characteristics as follows: {circle around (1)} It targets to the relevant tumor antigens with high specificity and affinity; {circle around (2)} It can bind monovalently to trigger factors on effector cells-cytotoxic cells and result in cross link only when BsAb binds to tumor antigens due to lack of Fc domain; {circle around (3)} BsAb is able to promote the effective cytotoxicity and inflammation selectively produced by the corresponding group of leukocytes at tumor sites; {circle around (4)} BsAb must be humanized to minimize induction of human anti-mouse response following repeated uses; Finally, {circle around (5)} BsAb should be not only small enough to penetrate into tumors but also large enough to keep in the circulation for a sufficient time.
Based on these points described above, numerous BsAbs triggering many kinds of immune effector cells and targeting different tumor cells have been developed in the past few years, wherein the effector cells include T lymphocytes, NK cells, monocytes, macropghages, neutrophils, LAK cells (lymphokine-activated cytotoxic cells) and TIL cells (tumor infiltrating lymphocytes) etc. T cells are commonly recognized as the major specific cells for immune responses. CD3 expressed on the surface of all mature T cells is the common surface marker for T cells. CD3 binds to TCR non-covalently, forming the whole TCR-CD3 complex, and involves in immune responses against antigen stimulus. Now CD3 is surface trigger molecule on immune effector cells used most widely and successfully. Following anti-CD3 antibody within BsAb binds to CD3 molecule on the surface of T cells, numerous effects as follows will be produced to kill tumor cells. These effects include: (1) proliferation and differentiation of T cells. Firstly, BsAb can activate the rest T cells, resulting in Th cell and Tc cell derived from the premature effector T cells with CD4+ or CD8+. Secondly, BsAb can activate numerous memory cells to proliferate and differentiate into effector T cells which will attack and kill tumor cells. The number of effector cells is directly related to the rate of tumor elimination. (2) release of cytokines: CD4+ Th cells activated by BsAb can secrete a great deal of IL-2. IL-2 not only stimulates the proliferation of Th cells in autocrine, but also activates naive CD8+ T cells in paracrine to become Tc cells, resulting in enlargement of cytotoxicity of Tc cells. In addition, IL-2 is a costimulating signal for activating T cells. Therefore, IL-2 plays a vital role in BsAb-mediated immune effects. Some other cytokines, such as TNF-α and IFN—Y are produced in the process of T-cell activation and can produce ‘stander-by’ effect by inhibiting the growth of ‘stander-by’ tumor cells through the medium among cells. (3) cytotoxicity: In vitro experiments indicate that mediated by BsAb, CD8+ Tc interacts with tumor cells directly, releases cytotoxic materials through granule exocytosis and lyses target cells, which takes place rapidly usually within 4-6 hours following targeting tumor cells. The major components in the cytotoxic materials are perforin and serine easterases or granzymes. Perforins can attack the plasma membrane and form ion channels, thus causing entry of plenty of ions and water, resulting in the lysis and necrosis of cells while granzymes are similar to lymphotoxin, capable of activating DNases in the cell, thus causing lysis of nucleic DNA, resulting in the apoptosis of target cells.
Currently, Fv fragment is widely used for construction of BsAb, since it is the minimal unit with the complete antigen-binding site, small (about ⅙ of the whole antibody), absence of Fc domain, lower immunogenicity, easily penetration into the wall of blood vessels and solid tumors, easily expressed in E. coli and lower production cost. However, Fv is unstable and easy to dissociate in vivo because the covalent bond between VH and VL domains is unable to generate. In order to improve the stability of Fv fragment, a polypeptide intralinker between VH and VL domains is used to form so called ScFv. The intralinker is commonly a short flexible peptide with 15 amino acid residues in length such as (Gly4Ser)3. In the present invention, the said intralinker was used in both ScFvs. As mentioned above, there are several methods to construct BsAb. In the present invention, we constructed the single-chain bispecific antibody (ScBsAb) connected by an interlinker. The general principle for designing interlinkers is to ensure the proper pairing and folding of variable domains from two antibodies, furthermore keep the biological activities and stability of the said antibody. In addition, the said interlinker should endow BsAb some novel properties, such as easy purification and prolonged half-life time in the plasma etc. Two kinds of interlinkers, Fc and HSA originally designed in the present invention as a useful provide a novel idea for designing interlinkers. 205c′ interlinker cited from literature was used to compare and verify the efficacy of interlinkers designed in the present invention and the value of the said design in construction of anti-ovarian BsAb. (1) Design of Fc interlinker: in order to minimize the immunogenicity and molecule size, small molecular antibodies are absent of Fv domains resulting in lack of several biological function, such as ADCC, CDC and the classic complement activating pathway. To resolve this problem, we devised the interlinkers to make up the said shortcoming of genetically engineered antibodies. IgG1 is the most potent molecule in inducing ADCC and CDC among four subtypes of IgGs. It can induce the classic complement activating pathway by combining to C1q with its C-terminal sequence of CH2, wherein Gly318, Lys320 and Lys322 sites locate in the surface of Fc molecule to form a cluster in conformation and combine to C1q directly. In addition, Asn297 of CH2 contains a glycosylation site which is vital to the effect of ADCC and CDC induced by Fc. Thus, a fragment from 297 to 322 of CH2 in human IgG was selected to construct the interlinker of ScBsAb. It has 26 residues in length and contains the glycosylation site Asn297, the C1q-binding site Glu318, Lys320 and Lys322 etc as well as an EcoRI site at the 5′ end and a SacI site at the 3′ end for the purpose of gene clone. ScBsAb constructed by this strategy is expected to have the prolonged half life time in vivo and the effect for inducing CDC similar to Fc. (2) HSA interlinker: Because of the smaller size, small molecular antibodies have fast renal clearance, which results in a short retention time in immunotherapy thus causing curative effects unperfect although the shorter half life time is benefit for immunoimaging diagnosis of tumors. Therefore, we devised HSA interlinker which is expected to prolong the half life time of ScBsAb in vivo, improve the stability and solubility of ScBsAb. HSA (human serum album) is an important component of human serum. It is widely used as a stable natural vector because of its stability, several week half-life time, lack of specific enzymatic and immunological activities and slow clearance in liver. It was showed in research that the stability of proteins fused with HSA increased 20 to 40 times in animals. HSA molecule with 585 amino acids in length is composed of three domains, wherein the third domain DIII alone possesses the vector function of the whole molecule. Herein, a fragment with 25 residues from 403 to 427 of DIII domain, which is lack of Cys but rich in polar amino acids in HSA was used as another interlinker in construction of BsAb to improve the stability and prolong the half-life time in vivo. (3) 205c′ interlinker: This interlinker is 25 amino acids in length devised by Gruber in construction of anti-TCRxanti-fluorescence scBsAb. The purpose of utilizing 205c′ interlinker was to compare and verify the efficacy of interlinkers designed in the present invention and the value of the said design in construction of anti-ovarian BsAb.
Facing HAMA problem induced by murine antibodies in clinic that strongly limits repeated use and dose, further causing the poor curative efficacy, murine antibodies must be humanized to minimize their heterology, which is the urgent affairs for preparation of antibodies used in clinic. The scFv against CD3 molecule in ScBsAb used in the present invention is a reshaped antibody through humanization. The reshaped antibody, so-called CDR-grafted antibody or humanized antibody, is constructed by grafting complementarity-determing regions (CDRs) from the variable domains of rodent antibodies into the framemork regions of human variable domains. The space structure of antigen-binding sites of antibodies is mainly determined by six CDRs of variable domains. The said CDRs form three loops, which have decisive effects in antigen-antibody recognition, in the upper site of variable domains supported by four β-sheet domains. The said reshaped antibody remains the antigen-binding ability as well as the most characteristics of human antibody, therefore minimizing HAMA response effectively.
Ovarian cancer remains the leading cause of death from gynecologic malignancies. The five-year survival rate maintains only 30%. Because of lack of the effective diagnostic methods for ovarian cancer located deep into pelvic cavity and the vague symptoms associated in the earlier stage, most patients with ovarian cancer present with an advanced stage of cancer. Although methods of surgical operation advance, drugs of chemotherapy renew and treatments of radiotherapy improve stepwise, the prognosis of ovarian cancer didn't improve at all. The easy recurrence after surgical operation and the side effects and drug tolerance after repeated use of chemotherapy strongly influence the effects of treatments. Therefore, specific diagnostic methods for earlier stage of cancer and the timely clearance of residual focuses are the key step for improving prognosis. BsAb against the related antigens of ovarian cells is regarded as powerful tools in clinic.